HELP! MY construct.. (ligation gel interpretation)
HARDIES at THORIN.UTHSCSA.EDU
HARDIES at THORIN.UTHSCSA.EDU
Wed Feb 1 10:55:44 EST 1995
Heidi Moss wrote:
> [having trouble putting an insert into Bluescript]...
> [She] Ran an agarose gel of vector alone--insert alone--ligation reaction
and hybridized: it seems that the ligation reaction was somehow
sub-optimal: the ligation band was fairly strong, but there were also
other bands/smear (that run BETWEEN the ligation band and insert band) in
addition to VERY FAINT unligated insert and vector. What are
I didn't fully understand the problem description, but this may help
you to interpret your diagnostic ligation gel. The product you are
trying to make is a closed circle of vector + insert. This circle
won't be highly supercoiled like the DNA you isolate from a gel, but
rather it will run more like a relaxed or nicked circle. It will be
spread into a ladder of bands because of the different winding numbers
in different molecules, and the exact position on the gel will be
sensitive to run temp., salt conc. in the buffer, etc., in comparison
to those parameters during the ligation itself. The ladder usually
looks more like a faint smear, because you've superimposed ladders from
various sized species caused by multiple insertion, no insertion, etc.
So an optimal ligation produces all smear and no bands.
Bands on the gel represent problems in the ligation. If any
insert band remains, then some portion of the insert is unligatable
*on both ends*. There should be no vector left either, unless you've
phosphatased it, in which case some will probably remain depending
on the vector:insert ratio. If you have a strong band running as a
linear fragment of the vector + insert length, or 2x the insert length,
this means that one end of the insert is ligating, but the other end
is unligatable. This is what I think you are calling the "ligation band".
Often this happens because the first enzyme you cut the
insert with was overdigested too much and damaged the end, but there
are a variety of other ways to get this problem. Other bands mapping
to sensible linear combinations similarly indicate a problem closing
some of the ends but not others. If you have a band that runs at
the high limit of the gel, then you are making concatemers rather than
circles because the concentration of DNA in the ligation is too high.
> 6) My mini preps always look weird -- many bands, most clones
were different from each other, some vector size, some shifted vector size,
some nowhere near the vector size, ALL with weird insert/vector
(digested appeared DIFFERENT than the undigested, and hybridization data
showed that none of the clones were what I wanted. What the heck was I
This follows from my suspicion that you are making linears consisting
of one vector joined to one insert. These linears transform at low
frequency and are circularized by illegitamate recombination to make
circles of unpredictable sizes. They are essentially versions of what
you tried to make with variable sized deletions of part of the insert
or part of the vector. Usually the deletion starts cleanly from the
problem junction and proceeds a variable distance into either the
vector or insert.
Hope this helps.
Steve Hardies, Assoc. Prof. of Biochem., Univ. of Texas HSC at San Antonio
Hardies at uthscsa.edu
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